Comparing Immune Profiles of Two Cell Lines: The Response to LPS of Murine RAW 264.7 and J774A.1 Macrophages.
Location
Turner Gymnasium
Access Type
Open Access
Entry Number
52
Start Date
4-5-2023 12:00 PM
End Date
4-5-2023 1:30 PM
College
Lynchburg College of Arts and Sciences
Department
Biomedical Science
Keywords
RAW 264.7, J774A.1, TNF-alpha, Murine macrophage, LPS, Immune response, Nitric Oxide, Growth curve, Inflammation
Abstract
Macrophages are significant to innate immunity and play a large role in inflammatory response initiation, maintenance and resolution. The RAW 264.7 macrophage line is widely studied and therefore a well vetted immunological model to compare the response of the less frequently used J774A.1 line. Macrophages are stimulated by bacterial products (pathogen associated molecular patterns or PAMPs), cellular damage, and other soluble mediators (cytokines). Inflammatory responses of macrophages can be measured by quantitating production of nitric oxide (NO) and cytokines released such as tumor necrosis factor-alpha (TNF-alpha) in response to PAMP bacterial lipopolysaccharide (LPS). Determination of growth rate in culture of J774A.1 macrophages was identified as necessary to evaluate response to LPS. A culture attempt to establish standard growth rate curve from pass 15 of J774A.1 cell line was developed, which returned a general doubling time (DT) of 18 hours compared to RAW 264.7 (24.1 hrs). Resulting DT allowed for corrections of NO response per 10,000 cells to evaluate growth rate differences as a confounding variable. Results suggest that RAW 264.7 cells respond more robustly to equivalent stimulation by LPS compared to J774A.1 cells in both NO and TNF-alpha response. When correcting for NO response per 10,000 cells, RAW 264.7 cells still show a greater responsiveness. In response to 100 ng/mL LPS stimulation, nitrite from RAW 264.7 cells was measured to be approximately 60 uM whereas nitrite measurement of J774A.1 was approximately 30 uM. Cytokine TNF-alpha measurements were a similar trend of greater responsiveness in RAW 264.7 cells at 100 ng/mL LPS (2560 pM) than J774A.1 (1995 pM). This data suggests RAW 264.7 to be a more vigorous model for examining inflammatory responses compared to J774A.1 macrophages.
Faculty Mentor(s)
David O. Freier MA, Ph.D
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Comparing Immune Profiles of Two Cell Lines: The Response to LPS of Murine RAW 264.7 and J774A.1 Macrophages.
Turner Gymnasium
Macrophages are significant to innate immunity and play a large role in inflammatory response initiation, maintenance and resolution. The RAW 264.7 macrophage line is widely studied and therefore a well vetted immunological model to compare the response of the less frequently used J774A.1 line. Macrophages are stimulated by bacterial products (pathogen associated molecular patterns or PAMPs), cellular damage, and other soluble mediators (cytokines). Inflammatory responses of macrophages can be measured by quantitating production of nitric oxide (NO) and cytokines released such as tumor necrosis factor-alpha (TNF-alpha) in response to PAMP bacterial lipopolysaccharide (LPS). Determination of growth rate in culture of J774A.1 macrophages was identified as necessary to evaluate response to LPS. A culture attempt to establish standard growth rate curve from pass 15 of J774A.1 cell line was developed, which returned a general doubling time (DT) of 18 hours compared to RAW 264.7 (24.1 hrs). Resulting DT allowed for corrections of NO response per 10,000 cells to evaluate growth rate differences as a confounding variable. Results suggest that RAW 264.7 cells respond more robustly to equivalent stimulation by LPS compared to J774A.1 cells in both NO and TNF-alpha response. When correcting for NO response per 10,000 cells, RAW 264.7 cells still show a greater responsiveness. In response to 100 ng/mL LPS stimulation, nitrite from RAW 264.7 cells was measured to be approximately 60 uM whereas nitrite measurement of J774A.1 was approximately 30 uM. Cytokine TNF-alpha measurements were a similar trend of greater responsiveness in RAW 264.7 cells at 100 ng/mL LPS (2560 pM) than J774A.1 (1995 pM). This data suggests RAW 264.7 to be a more vigorous model for examining inflammatory responses compared to J774A.1 macrophages.